Developmental choices in mammalian hematopoietic cells depend on the balance among the expression levels of tightly regulated transcription factors. Extremely accurate regulation of these genes is not only critical for normal hematopoietic development but also to avoid leukemia. In terms of mechanism, the question is what DNA-protein interactions determine the level of expression of each transcription factor itself. PU.l in particular has a complex pattern of expression in different hematopoietic lineages that is crucial for normal differentiation. Qualitative as well as quantitative differences in PU.l expression pattern distinguish myeloid and T-lymphocyte developmental pathways. This could indicate that PU.l depends on a variety of different regulatory sequences to activate or repress it in different cellular contexts, as hinted by our preliminary data. However, in mammalian hematopoiesis there has not been any adequate system to map the functional roles of any potential regulatory sequence of interest across multiple hematopoietic cell types. This proposal is to develop a new model system for these analyses that should enable the same clonal integration of a regulatory sequence-reporter construct to be monitored in diverse hematopoietic lineages on a relatively high-throughput basis as compared to transgenesis. We will exploit a new in vitro differentiation system that enables embryonic stem cells (ES cells) to develop directly into both lymphoid and erythromyeloid cells. BAG and plasmid regulatory-sequence reporter constructs will be transfected into undifferentiated ES cells and then tested for activity as those cells differentiate in distinct developmental pathways. We plan to demonstrate the effectiveness of this system by using it to map the combined positive and negative regulatory elements of the PU.l (Sfpil) gene that govern its divergent patterns of expression in T-lymphoid precursors as contrasted with myeloid precursors. Our specific aims are: (1) to optimize the ES cell system for analysis of reporter expression in the myeloid and lymphoid lineages; (2) to use this system to define the cis-regulatory elements that control the pattern of PU.l expression in T-lineage development; and (3) to verify these conclusions by analysis of reporter expression in mice derived from the ES cell transfectants. Summary: What regulates the regulators? Here we propose to develop a new approach to this important question, using a very recent advance in cultures of mouse embryonic stem cells to overcome past technical barriers. This should permit faster and cheaper experiments to reveal how powerful regulatory genes are themselves controlled. [unreadable] [unreadable]